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Mouse muscle protein expression during aging and calorie restriction - Analysis utilizing SILAC mouse
S56 1-wk adaptation, 21-day 6 -head-down-tilt bed rest,1-wk recovery. In the first phase, 4 over 8 subjects received daily potassium bicarbonate supplementation whereas the second phase involved the subject group crossover. Before and after each bed rest, we assessed: (I) body composition by bioimpedance; (II) whole-body protein metabolism and (III) glutathione fractional-synthesis-rate (GSH-FSR) in erythrocytes by stable isotopes, using [ring-2H5]-phenylalanine, [2H2]-tyrosine and [2H2]-glycine primed-continuous infusions as well as single bolus of [ring-2H4]-tyrosine. Results: Fat-free mass decreased (-3.60.5%, p<0.001) after unloading. Inactivity decreased protein synthesis (-62%; pZ0.03), increased phenylalanine hydroxylation (an index of net protein loss) (+205%; p<0.01) whereas did not modify protein degradation. There was significant effect of bicarbonate supplementation in preventing bed rest-mediated acceleration of phenylalanine hydroxylation (ANCOVA; p<0.05). Total GSH, GSSG concentration and GSH FSR in erythrocytes did not change following unloading. Bicarbonate supplementation in bed rest increased total GSH (+52%; pZ0.04), decreased GSH FSR was (-3813%; pZ0.04) and did not change GSSG concentration. Conclusions: Bicarbonate supplementation counteracts bed rest-induced negative effects on protein metabolism and decreases glutathione utilization leading to increased glutathione availability. MOUSE MUSCLE PROTEIN EXPRESSION DURING AGING AND CALORIE RESTRICTION - ANALYSIS UTILIZING SILAC MOUSE Piotr Zabielski *, Ian R. Lanza *, Carrie J. Holtz Heppelmann $, Daniel R. Jakaitis *, Benjamin J. Madden $, H. Robert Bergen$, K. Sreekumaran Nair * * Division of Endocrinology and Metabolism, Mayo Clinic College of Medicine, Rochester, MN, USA
Abstracts $
Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN, USA
Calorie restriction has been shown to delay senescence, aging-associated diseases and prevent age-related decrease of muscle oxidative capacity but the underlying mechanism remains to be fully defined. Since previous findings demonstrated alterations in protein turnover we used muscle tissue from fully labeled mice (SILAC mice) combined with mass spectrometry to elucidate impact of calorie restriction on muscle protein expression patterns in B6D2F mice. Young animals (YAL) of 8 months old were fed ad libitum, whereas old animals (OAL) of 24 months old were either fed ad libitum (OAL) group -) or calorie restricted (OCR) (kcals reduced to 60% of OAL). Gastronomies muscle samples from YAL, OAL, and OCR (nZ3 per group) were mixed 1:1 per protein content with SILAC mouse muscle (ISTD mouse, DBA2H strain, 98% MPE labeling with heavy isotope 13C6-Lysine). Combined samples were resolved by 1DGE and analyzed by nanoLC-LTQ-ORBITRAP mass spectrometry. Light vs. heavy 13C6-lysine ratios of 7524 peptides from 876 proteins were used to asses relative protein concentration between experimental groups. Compared to YAL, OAL animals displayed up-regulation of several muscle structural proteins, enzymes involved in b-oxidation, ketone body metabolism, iron uptake and protein degradation, whereas fatty acids synthesis, mitochondrial respiratory chain proteins and protease inhibitors were down regulated. In contrast, OCR animals displayed up regulation of several mitochondrial respiratory chain proteins and those involved in anti-oxidant defense, fatty acid synthesis, ketone body metabolism, DNA stability and proper protein folding, whereas down regulated protease inhibitors, metalloproteinase activators and iron uptake/transport proteins as compared to OAL counterparts. It is concluded calorie restriction in aging mice affects primarily proteins involved in fuel selection and energy and protein metabolism.
Abstracts $
Mayo Proteomics Research Center, Mayo Clinic College of Medicine, Rochester, MN, USA
Calorie restriction has been shown to delay senescence, aging-associated diseases and prevent age-related decrease of muscle oxidative capacity but the underlying mechanism remains to be fully defined. Since previous findings demonstrated alterations in protein turnover we used muscle tissue from fully labeled mice (SILAC mice) combined with mass spectrometry to elucidate impact of calorie restriction on muscle protein expression patterns in B6D2F mice. Young animals (YAL) of 8 months old were fed ad libitum, whereas old animals (OAL) of 24 months old were either fed ad libitum (OAL) group -) or calorie restricted (OCR) (kcals reduced to 60% of OAL). Gastronomies muscle samples from YAL, OAL, and OCR (nZ3 per group) were mixed 1:1 per protein content with SILAC mouse muscle (ISTD mouse, DBA2H strain, 98% MPE labeling with heavy isotope 13C6-Lysine). Combined samples were resolved by 1DGE and analyzed by nanoLC-LTQ-ORBITRAP mass spectrometry. Light vs. heavy 13C6-lysine ratios of 7524 peptides from 876 proteins were used to asses relative protein concentration between experimental groups. Compared to YAL, OAL animals displayed up-regulation of several muscle structural proteins, enzymes involved in b-oxidation, ketone body metabolism, iron uptake and protein degradation, whereas fatty acids synthesis, mitochondrial respiratory chain proteins and protease inhibitors were down regulated. In contrast, OCR animals displayed up regulation of several mitochondrial respiratory chain proteins and those involved in anti-oxidant defense, fatty acid synthesis, ketone body metabolism, DNA stability and proper protein folding, whereas down regulated protease inhibitors, metalloproteinase activators and iron uptake/transport proteins as compared to OAL counterparts. It is concluded calorie restriction in aging mice affects primarily proteins involved in fuel selection and energy and protein metabolism.